Plasma Membrane Proteins PDF

Module 2: Plasma membrane, membrane proteins Slide 2: After this module, you should be able to describe how proteins are arranged and anchored in the lipid bilayer, describe the types of protein movement in the plasma membrane, explain how detergents solubilize membrane proteins, understand how red...

Module 2: Plasma membrane, membrane proteins Slide 2: After this module, you should be able to describe how proteins are arranged and anchored in the lipid bilayer, describe the types of protein movement in the plasma membrane, explain how detergents solubilize membrane proteins, understand how red blood cell ghosts are used to determine the positioning of membrane proteins, discuss the composition and role of the cell glycocalyx, explain the connection between glycosylation and blood groups, and describe the different functions of membrane proteins. Slide 3: Proteins constitute ~ 50 % of the membrane mass (range: 25% to 75%). The positioning of the proteins in the lipid bilayer depends upon the nature of the protein. Frequently, the membrane proteins are amphipathic; they have hydrophilic and hydrophobic parts. Such proteins may cross the bilayer and are called “transmembrane.” In the figure, the transmembrane proteins cross the bilayer as a single α helix; some of these proteins may have a covalently attached fatty acid inserted in the lipid layer (1). The transmembrane proteins could consist of multiple α helices (2) or form a rolled-up β sheet (β-barrel, 3). Some proteins are associated with the membrane via different attachments. A protein could be anchored to the cytosolic surface by an α helix (4). A protein can be also attached to the bilayer by a covalently attached lipid (5). A protein can be attached to the lipid chain via an oligosaccharide linker (6). There are also proteins attached to the membrane only by noncovalent interactions with other membrane proteins (7,8). Integral proteins are proteins that are permanently attached to the membrane, either by being transmembrane or associated with only one side of the membrane. Therefore, “integral protein” is a more inclusive term than “transmembrane protein.” Slide 4: The transmembrane proteins have intracellular and extracellular domains, separated by (trans)membrane domains. The transmembrane portions of the proteins adopt one of two tertiary structures: α helices or β barrels. The transmembrane domains are mostly composed of amino acids with nonpolar side chains. In these domains, all peptide bonds (polar!) form hydrogen bonds with one another. Such interactions are maximized in an α helix. Alternatively, multiple transmembrane segments of a polypeptide chain could be arranged as a β sheet in the form of a closed barrel. Such β barrel proteins are abundant in the outer membrane of mitochondria, and some of them form water-filled channels. In barrel-like structures, the side chains of the amino acids are localized according to polarity. Non-polar (hydrophobic) amino acids associate directly with the lipid bilayer and polar (hydrophilic) amino acids are localized internally (in the channel), facing the aqueous environment. Slide 5: Some proteins rely on anchors to associate with the membrane. One example is the glycosylphosphatidylinositol (GPI) anchor, consisting of a glycolipid attached to the carboxy-terminus of a protein (all proteins have an amino terminus and a carboxy-terminus). The glycolipids are in the outer leaflet, and as depicted in the figure, the GPI-anchored protein is on the exterior side. GPI-linked proteins might be preferentially located in lipid rafts. The two fatty acids of the glycolipid anchor the structure to the cell membrane. Other anchors are the fatty acid chains and prenyl groups, and these are mostly on the cytosolic face of the membrane. Slide 6: The membrane proteins fulfill different functions. They connect and join cells through cell junctions. Proteins attach the cell’s cytoskeleton to the extracellular matrix through junctional complexes. The proteins also function as “barcodes” (labels) that allow for cell recognition. Additional groups of proteins serve as receptors in signaling pathways, enzymes, or mediators of transmembrane transport. Slide 7: Similar to the lipid molecules in the membrane bilayer, proteins move within the membrane. Membrane proteins rotate about an axis perpendicular to the bilayer and this movement is designated as “rotational diffusion”. Proteins also move laterally, and this type of movement is “lateral diffusion”. There is experimental evidence for protein movement. In this experiment, mouse and human cells were fused to produce hybrid cells. Antibodies that distinguish between mouse and human membrane proteins were added to the hybrids. At the beginning of the labeling period, the green fluorescence that labeled the mouse cell surface proteins was segregated from the red fluorescence that labeled the human cell surface proteins. However, within 40 minutes, the two sets of proteins exhibited a mixed pattern on the cell surface. The lateral diffusion of proteins can be measured with a technique called “fluorescence recovery after photobleaching” (FRAP). In this procedure, the protein of interest is labeled with a fluorescent group. Then the fluorescence is bleached in a small area and the time taken for the adjacent membrane proteins with unbleached ligand to diffuse into the bleached area is measured. Slide 8: This video demonstrates FRAP: https://www.youtube.com/watch?v=CfRvmtBdZ9I Slide 9: Fluorescence recovery after photobleaching (FRAP) also detects membrane proteins that are not moving. One way by which the movement of membrane proteins is restricted is through assemblies of many molecules inside or outside the cell. This could be an association with the cytoskeleton (inside the cell) or the extracellular matrix (outside the cell). The image on the right is of an epithelial cell with an apical and basolateral domain that constrict the movement of the membrane proteins. Such partitioning allows for the directional transport of molecules - in this case, from the intestinal lumen into the blood circulation. The separation between apical and basolateral domains is due to the formation of tight junctions between the cells. Another example is the restricted movement of membrane proteins in neurons. In neuronal cells, one membrane domain encloses the cell body and dendrites, and another membrane domain encloses the axon. Slide 10: How do we know what we already know about membrane proteins? There are approaches, via which membrane proteins are isolated, separated from lipids, solubilized, and characterized. First, we need to disrupt the hydrophobic associations in the lipid bilayer. This separation is accomplished with detergents that are amphiphilic (amphipathic) molecules forming micelles in water. The hydrophobic ends of detergents bind to the hydrophobic regions of the membrane proteins, displacing the lipid molecules. The polar ends of the detergents bring the proteins into the solution as detergent-protein complexes. Two types of detergents are shown: the hydrophilic ends of detergents can be either charged (ionic), as in sodium dodecyl sulfate (SDS) that has a sulfate group, or uncharged (nonionic), as in the Triton detergent. Such hydrophilic groups form hydrogen bonds with the water molecules. The second image illustrates the ability of the detergents to form micelles. The “Critical Micelle Concentration” or CMC of a detergent is the concentration of a detergent, at which micelles start to form. Slide 11: Another tool to study the membrane proteins are the red blood cell ghosts. The term refers to empty red blood cell membranes. Why do we use red blood cells? We do so because mature red blood cells do not have nuclei and internal organelles. Their cell plasma membrane is the only membrane in the cells. The ghosts are prepared by exposing the cells to salt concentrations lower than those in the cell interior. This type of solution is hypotonic. This treatment results in water flowing into the cells, followed by cell lysis and the release of cytosolic proteins. The plasma membrane is then re-sealed and various experiments could be performed. For example, the question of where a membrane protein is positioned (on which side of the membrane) is answered by using a label such as a fluorescent marker that is water-soluble and therefore, does not penetrate the lipid bilayer. Such marker attaches covalently to the portion of the protein that is exposed on the surface of the cell ghosts. After labeling, the membranes are solubilized with a detergent, and the proteins are separated by a technique called SDS polyacrylamide-gel electrophoresis. Alternatively, the external or internal surface is exposed to proteolytic enzymes, which do not cross the membrane. If a protein is partially digested from both surfaces, it must be a transmembrane protein. Slide 10: Through a series of experiments with red blood cell ghosts, three major cell membrane proteins have been identified. The first of the proteins is spectrin. Spectrin is a peripheral protein on the cytosolic side of the membrane. It forms heterodimers, which assemble into tetramers. The tetramers are linked to actin and other cytoskeletal proteins to form a meshwork under the membrane. This additional spectrin cytoskeleton in the red blood cells imparts strength to mechanical stress. The red blood cells are forced through narrow capillaries, and therefore, they experience extreme mechanical stress. The spectrin-actin association is also important in maintaining the biconcave shape of erythrocytes which maximizes the amount of hemoglobin and oxygen carried by each red blood cell. The second protein identified using red blood cell ghosts is glycophorin. This is a single-pass transmembrane protein, usually a homodimer. Its hydrophilic amino terminus carries a carbohydrate. The third protein is Band 3. This is a multi-pass transmembrane protein. It functions as an anion transporter that allows the passage of bicarbonate (HCO3-, out) in exchange for chloride (Cl-, in). This transport results in increased levels of carbon dioxide (CO2) delivered by the blood to the lungs. Slide 11: The membrane proteins could be modified. Most of the transmembrane proteins are glycosylated. The sugar residues are added inside the lumen of the endoplasmic reticulum and Golgi apparatus. The oligosaccharides on the membrane proteins are always on the non-cytosolic (external) side of the membrane, and such glycosylated proteins frequently function as cell surface receptors. Another distinction between the exterior and interior parts of the transmembrane proteins is that the sulfhydryl groups in the cytosolic domain do not form disulfide bonds because the reducing environment in the cytosol maintains these groups in their reduced (-SH) form. In contrast, in the extracellular domain, such groups form disulfide bonds. Slide 12: The glycocalyx is a carbohydrate-rich zone on the cell surface. It can be visualized by stains or lectins labeled with a fluorescent dye. The lectins are proteins that bind carbohydrates. The glycocalyx is composed of glycoproteins, glycolipids, and proteoglycans. Figures A and B demonstrate the differences between glycoproteins and proteoglycans. Whereas glycoproteins are proteins with short and highly branched glycan chains, the proteoglycans have a core protein with a great number of glycosaminoglycan (GAG) chain(s) attached (so, proteoglycans are a subgroup of glycoproteins). The carbohydrate chains are long and negatively charged under physiological conditions. The glycocalyx protects cells against mechanical and chemical damage. It also keeps foreign objects and cells at a distance, prevents some protein-protein interactions, binds antigens, affects transplant compatibility, and impacts fertilization. Slide 14: Putting it together. Polypeptide chains cross the membrane bilayer as a single α helix or multiple times, as a series of α helices, or as a β sheet (“barrel”). Other proteins, however, can attach to either side of the membrane; the bond can be noncovalent (binding to transmembrane proteins) or covalent (binding to lipids). Membrane proteins function as receptors, enzymes, transport proteins, anchors, and “labels” that facilitate recognition. Membrane proteins can diffuse in the membrane. Some proteins, however, are immobilized. On the outer lipid monolayer, the proteins and lipids may have oligosaccharide chains to form the glycocalyx, a carbohydrate-rich zone that protects the cell from mechanical and chemical damage. Supplementary Materials Spectrin is a component of the protein network that covers the cytoplasmic surface of vertebrate erythrocyte membranes. It is composed of two subunits, alpha (α) and beta (β). The spectrin molecules are extended and flexible, with actin-binding domains at each end. The α and β subunits associate laterally to form antiparallel heterodimers. The resulting heterodimers are assembled head-head to form heterotetramers. The spectrin-actin network in erythrocytes is coupled to the membrane bilayer primarily through the association of spectrin with ankyrin, which in turn is bound to the cytoplasmic domain of an anion exchanger. From https://www.sigmaaldrich.com/US/en/technical- documents/technical-article/research-and-disease-areas/cell-signaling/spectrin. Are you acquainted with the Human Protein Atlas? Here is the immunofluorescent detection of spectrin from the Atlas: (image on the left: spectrin in green; microtubules in red; nuclei in blue; the image on the right. spectrin in green).  Would you like to explore the Atlas? You could find more about Band 3 (also called SLC4A1) and glycophorin.